Using molecular, biochemical and structural approaches, over several years, we have contributed to defining how specific human BER proteins recognize and process target lesions, as well as coordinate with other components of the pathway. This research has centered largely on apurinic/apyrimidinic endonuclease 1 (APE1), the major mammalian protein for repairing abasic sites in DNA, and x-ray cross-complementing 1 (XRCC1), a non-enzymatic scaffold protein that facilitates the efficient execution of single-strand break (SSB) repair. Our most recent work has found that (i) APE1 can endonucleolytically cleave RNA substrates in its role to cleanse the transcript pool and regulate gene expression via a reaction mechanism similar to that used on AP-DNA;(ii) XRCC1 supports an emerging genomic maintenance pathway termed replication-associated BER in part through a physical association with UNG2, the major nuclear uracil DNA glycosylase;(iii) the protein activation-induced cytosine deaminase (AID) generates uracil DNA intermediates in immunoglobulin gene variable and switch regions as part of the process to create antibody diversity;and (iv) XRCC1 coordinates disparate responses and multi-protein repair complexes that are dependent on the nature and context of the DNA damage. APE1 is the major mammalian enzyme responsible for the repair of abasic sites in DNA, and has functions in SSB repair and other cellular processes, including transcriptional regulation. We are in the process of establishing genetically modified cell lines to dissect out the precise contribution of each proposed function of APE1 (i.e. its nuclease activity, redox regulatory role, etc.) in cell growth/viability, genome maintenance, and protection against DNA-damaging agents. The underlying hypothesis is that APE1, and BER more generally, plays a critical role in dictating cellular responsiveness to genotoxic insults and susceptibility to disease manifestation, particularly in the face of certain environmental exposure(s). Defining which of the many reported functions of APE1 are critical to normal cellular activity is a key step towards understanding the potential relationship of the protein to disease risk. DNA interstrand crosslinks (ICLs) can arise from reactions with endogenous chemicals, particularly the lipid peroxidation product malondialdehyde, as well as from exposure to a variety of clinical anti-cancer agents, such as bifunctional alkylators. Recent studies suggest a role for BER in the processing of these highly toxic DNA intermediates, which are absolute blocks to transcription and replication, and thus, lethal adducts. We are performing experiments using developed strategies, e.g. laser microirradiation coupled with high resolution confocal microscopy, to define the molecular involvement of specific BER factors in ICL removal. Elucidating the roles of BER proteins in ICL resolution may reveal new targets relevant to anti-cancer treatment paradigms. XRCC1 is a non-enzymatic scaffold protein that appears to orchestrate most DNA repair responses at sites of SSBs. Our efforts aim to evaluate the contributions of XRCC1 to DNA damage responses, genomic stability and telomere maintenance in defined mammalian cell lines. Our hypothesis is that defects in SSB repair will be more deleterious to non-dividing, terminally-differentiated cells (e.g. neurons) than dividing cells, because dividing cells repair SSBs not only by SSB repair, but also by replication-mediated homologous recombination. Such studies on XRCC1 will go a long way towards elucidating the molecular mechanisms underlying cancer, aging and neurodegenerative disease.